Our ultimate goal is elucidation of factors, primarily genetic, that control cellular differentiation in a higher eukaryote, Drosophila. For this, the alpha-amylase system was chosen for analysis. Structure and function of Amy, the structural gene for amylase, are under study in our attempt to unravel the mechanisms that regulate each step from transcription to the final phenotypic expression of this gene. A system of "temporal" control genes for tissue specific expression of Amy in the midgut is being analyzed. Cis- and trans-acting elements are being genetically defined so their mode of action may be explored at the molecular level. The approaches of genetics, biochemistry, cytology and molecular biology are used. The project for 1981 includes: (1) in situ hybridization to salivary chromosomes of 3H-cRNA made against a clone, pDmG3F9, that apparently contains all of part of Amy from D. melanogaster inserted into the pBR322 plasmid of E. coli; (2) analysis of the primary structure of DNA in the pDmG3F9 insert by (a) restriction mapping and sequencing of fragments, (b) screening of lambda phase libraries of D. melanogaster fragments for additional and overlapping clones complementary to our probe, and (c) restriction mapping and sequencing of secondary clones, including flanking regions of Amy for clues to control elements; (3) mutagenic experiments utilizing a selective genetic screen for amylase deficient mutants (deficiencies, "null", control factors for no midgut activity); (4) recombinant second chromosome viability tests to study potential epistasis between Amy and adp loci, and to locate and size genetic regions affecting viability in order to identify the best chromosome 2 for recombination and population experiments; (5) continued analysis of temporal control genes, e.g. map; and molecular characterization of pure amylase allozyme, including N-and C-terminal identification, peptide mapping and the start of amino acid sequencing.